DNA Fingerprinting Please highlight all your answers with a yellow background or use a different color font that is easy to read LEARNING OBJECTIVES 1 Describe the structure and function of DNA 2 Describe the procedure of agarose gel electrophoresis and its application to DNA separation 3 Perform selective DNA digestion using restriction enzymes 4 Separate the DNA by electrophoresis and estimate the size of a DNA fragment 5 Evaluate and interpret various scenarios by which DNA fingerprinting is used as a biotechnology tool to identify individuals and show whether individuals or animals are related to each other INTRODUCTION This experiment introduces the basic concepts of DNA fingerprinting a method used in various medical and forensics procedures as well as in paternity determinations This protocol will demonstrate the similarities and differences in organisms at the genetic level The basic concept of any DNA fingerprinting protocol includes the extraction of DNA from any cell the restriction or cutting of the DNA by enzymes called endonucleases and the analysis of the resulting DNA fragments using agarose gel electrophoresis Two basic techniques exist that allow analysis of DNA Polymerase Chain Reaction PCR is commonly used in molecular and genetic research Restriction Fragment Length Polymorphism RFLP analysis is another technique used to compare differences and similarities between individuals at the genetic level We will use this second technique in our investigation Many forensic scientists including those involved with criminal investigations use RFLP analysis in order to eliminate potential suspects related to a crime scene For our experiment you will be given five DNA samples consisting of DNA collected from a crime scene and DNA collected from five different suspects Your task will be to identify the true criminal using the RFLP analysis technique DNA FINGERPRINTING RFLP ANALYSIS Each person has similarities and differences in their DNA sequences This means that if we look at a population of people we will see variation in the sequence of nucleotides of a particular locus on a chromosome RFLP analysis uses this variation as a method to identify individuals based on their particular variation The size of the fragments is reflected by the variations in individuals DNA AGAROSE GEL ELECTROPHORESIS Electrophoresis separates DNA fragments according to their relative size DNA fragments are loaded into an agarose gel which is placed into a chamber filled with a conductive liquid buffer solution Tris Acetic Acid EDTA or TAE See Figure 1 Figure 1 Electrophoresis chamber RESTRICTION DIGESTION OF DNA Because they cut DNA restriction enzymes are the chemical scissors of the molecular biologist When a particular restriction enzyme recognizes a particular four or six base pair hp recognition sequence on a segment of DNA it cuts the DNA molecule at that point This can leave blunt ends AluI for example or sticky ends HindIII In some cases this is done so that researchers can insert a new piece of DNA into the sequence In our case we will be cutting the DNA into various sizes in order to match the crime scene DNA with a suspect Both of those DNA samples should have the same size pieces when we run our gel See Figure 2 for examples of enzyme recognition sites Figure 2 Restriction digestion of DNA restriction Figure 3 DNA restriction fragments in agarose gel Agarose is a natural substance that upon being dissolved in liquid will solidify into a matrix like arrangement This matrix creates channels through the gel in which small molecules can pass A direct current is passed between wire electrodes at each end of the chamber causing a current to pass through the gel from one end to the other DNA fragments are negatively charged and when placed in an electric field will be drawn toward the positive pole and repelled by the negative pole The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than large ones Over a period of time smaller fragments will travel farther than larger ones Fragments of the same size will stay together and migrate in single bands of DNA Figure 3 shows the product after electrophoresis of Lambda DNA that has been digested with the HindIII restriction enzyme ACTIVITY 1 PRACTICE PIPETTING Your lab instructor will show you how to use a micropipette see Figure 4 It is very important that you pay close attention to this and that all people at your table practice this skill Be sure to always use a tip when pipetting Pipetting can be difficult particularly when pipetting such small portions Use the yellow tips and the container of colored water or loading dye for practice Practice adjusting the pipettor settings Do not turn to a setting below or above its range Use the practice loading dye to see what 5 L 10 L and 20 L look like in the tip It is important to be able to accurately measure each sample Using too little sample will result in minimal digestion Using too much sample will result in not having enough components to complete all of the tubes at your table Figure 4 Micropipettor https www jove com video 2754 aseptic laboratory techniques volume transfers with serological ACTIVITY 2 RESTRICTION DIGESTION OF DNA SAMPLES PROCEDURE 1 Make sure that the following materials are present at your lab table prior to beginning the lab DNA Electrophoretic unit with Gel tray 8 well comb Gel casting stand Sterile pipette tips Pipette 0 5 10 L Foam rack for 1 5 mL bullet tubes see Figure 5 Ice bucket with the following EcoRI Pstl Enzyme Mix labeled ENZ 1 tube 15 L Loading Dye LD 1 tube 2 Sharpie markers 600 mL beaker for used tips and tubes Colored water for pipetting practice Figure 5 Bullet tubes https www dreamstime com illustration eppendorf tube html 2 On the side counter you should be able to locate an ice bucket with ice containing 1 tube of each of the following Crime scene DNA Suspect DNA 1 2 3 4 and 5 CS crime scene SI suspect 1 3 Label the colored bullet tubes with the appropriate label indicating the DNA samples NOTE Label the cap of the bullet tube Green Blue Orange Violet Red pink Yellow 4 Locate the DNA samples on the side counter and transfer 10 L of S2 suspect 2 S3 suspect 3 S4 suspect 4 S5 suspect 5 each sample from the stock tube into the correspondingly labeled colored bullet tube Make sure the sample is transferred to the bottom of the tube To avoid confusion your TA may move these stock tubes from table